ES2995407B2 - Use of a Bacillus mycoides strain for the control of Xylella fastidiosa - Google Patents

Description

[0001] DESCRIPTION

[0003] Use of a Bacillus mycoides strain for the control of Xylella fastidiosa

[0005] The present invention belongs to the technical field of agricultural biotechnology, and relates to the use of i) a bacterial strain belonging to the family Bacillaceae, ii) the supernatant of a culture of a bacterial strain belonging to the family Bacillaceae, or iii) a composition comprising i) and/or ii) for the control of Xylella fastidiosa in a plant and/or in an insect vector. Furthermore, the present invention relates to a method for the control of X. fastidiosa in a plant, and a method for the control of X. fastidiosa in an insect vector.

[0007] BACKGROUND OF THE INVENTION

[0009] The Mediterranean region is one of the most important agricultural production areas in the world. However, this region faces an unprecedented threat from the emerging plant pest caused by Xylella fastidiosa, which is capable of causing the death of the plant it infects. X. fastidiosa affects more than 600 plant species, including economically important crops such as olive, grape, and citrus (Delbianco et al., (2022) EFSA Journal 20; Sicard et al., (2018) Annu Rev Phytopathol 56, 181–202).

[0011] Therefore, considerable research effort has been devoted to finding effective ways to combat X. fastidiosa and fight outbreaks of this bacterium in different crops. Current strategies include the use of antibiotics, bioactive compounds to boost plant resistance, bacterial and/or fungal strains, as well as metal-based formulations (EFSA Panel on Plant Health, 2016). Initial studies showed promising results for mineral-based formulations (e.g., zinc, copper, calcium) or chemical compounds such as citric acid or coated fosetyl-aluminum nanocrystals, but ultimately with limited success (Morelli et al., (2021) Microorganisms vol. 9 Preprint; Scortichinie et al., (2018) Phytopathol Mediterr 57, 48–72). Other treatments focused on the use of natural compounds such as detergent mixtures, natural oils and extracts (Bruno et al., 2021)L. Crop Protection 139).

[0013] However, to date, no effective field treatment is available to cure infected plants and eliminate X. fastidiosa, highlighting the urgent need for new control strategies. Given the European Union's commitment to advancing a sustainable food strategy through legislative initiatives (such as the EU Green Deal) focused on reducing the use of chemical pesticides in food production, it is necessary to provide new, effective, and environmentally friendly strategies for the control of X. fastidiosa in plants.

[0015] DESCRIPTION OF THE INVENTION

[0017] According to the results obtained by the present inventors, the cultivable sporobiota of the olive tree comprises different bacteria belonging to the family Bacillaceae, specifically bacteria of the genus Bacillus and Peribacillus, among which it was found that certain strains of the clade of B. subtilis, P. simplex and B. cereus presented antimicrobial activity against X. fastidiosa (Fig. 1).

[0019] The olive sporobiota refers to the set of bacteria capable of producing endospores that are found in the tissues of olive plants (either on their surface or within the tissues themselves), or in the soil where these plants grow. Thus, the cultivable olive sporobiota refers to those bacteria of the olive sporobiota capable of growing in culture media.

[0021] The present inventors found that the strains with antimicrobial activity against X. fastidiosa were Bacillus velezensis CECT 30858, Bacillus subtilis CECT 30859, Bacillus mojavensis CECT 30857, Peribacillus simplex CECT 30856, and Bacillus mycoides CECT 30855, unlike other bacterial strains tested also derived from the cultivable sporobiota of the olive tree. Therefore, the strains Bacillus velezensis CECT 30858, Bacillus subtilis CECT 30859, Bacillus mojavensis CECT 30857, Peribacillus simplex CECT 30856, and Bacillus mycoides CECT 30855 constitute an alternative to chemical pesticides for the control of X. fastidiosa in plants.

[0023] Thus, in a first aspect, the present invention relates to the use, henceforth referred to as the “use of the invention”, of:

[0024] i) a bacterial strain belonging to the family Bacillaceae,

[0025] ii) a supernatant from a culture of a bacterial strain belonging to the family Bacillaceae, or

[0026] iii) a composition comprising i) and/or ii),

[0027] for the control of Xylella fastidiosa in a plant and/or in an insect vector, wherein the bacterial strain belonging to the Bacillacea family in i) and ii) is selected from the list consisting of Bacillus mojavensis CECT 30857, Bacillus velezensis CECT 30858, Bacillus subtilis CECT 30859, Peribacillus simplex CECT 30856 and Bacillus mycoides CECT 30855.

[0029] The bacterial strains described in the present invention were isolated from the cultivable sporobiota of olive trees. The strains Bacillus subtilis CECT 30859 and Bacillus mojavensis CECT 30857 were isolated from endophytic samples of olive leaves grown in the municipality of Linares in Jaén (Spain), and these strains were received on June 1, 2023 at the Spanish Type Culture Collection, CECT (address: Parc Científic de la Universitat de València, Carrer del Catedràtic Agustín Escardino Benlloch, 9, 46980 Paterna, Valencia, Spain), with the University of Jaén as the depositor. The strains Peribacillus simplexCECT 30856 and Bacillus mycoidesCECT 30855 were isolated from soil samples from olive crops in Malaga, Spain, while the strain Bacillus velezensisCECT 30858 was isolated from soil samples from olive crops in the municipality of Linares in Jaen (Spain), and these strains were received on June 1, 2023 at the Spanish Collection of Type Cultures, CECT (address: Parc Científic de la Universitat de València, Carrer del Catedràtic Agustín Escardino Benlloch, 9, 46980 Paterna, Valencia, Spain), with the depositor being the University of Jaen.

[0031] Bacillus subtilis CECT 30859 and Bacillus mojavensis CECT 30857 are endophytic or endosymbiotic bacteria that live within a plant for at least part of their life without causing apparent disease, and they also share the same ecological niche as X. fastidiosa. Plant endophytic bacteria have been the subject of considerable attention for their ability to control diseases and insect infestations.

[0033] The present inventors have not only demonstrated the antimicrobial capacity of the bacterial strains cited in the present invention against aX. fastidiosa, but have also demonstrated the antimicrobial capacity of the supernatant resulting from the culture of the same against aX. fastidiosa.

[0034] In the present invention, the term “culture supernatant” refers to the liquid fraction of a liquid culture medium, preferably liquid, in which bacteria are cultivated, wherein the liquid fraction is separated from the bacteria by any means known to a person skilled in the art, such as centrifugation or filtration. The term “culture medium” refers to an aqueous solution containing the nutrients necessary for cell growth and/or survival, which can be used to culture cells for an extended period of time. Typically, cell culture media include the following components: an energy source, which is usually a carbohydrate compound, preferably glucose; amino acids, preferably the basic set of amino acids, including all essential and non-essential amino acids; vitamins and/or other organic compounds required in low concentrations; free fatty acids; and inorganic compounds, including trace elements, inorganic salts, buffer compounds, and nucleosides and bases.

[0036] Therefore, both the bacterial strains of the Bacillaceae family described in the present invention and the supernatant of their cultures can be used in methods of control of X. fastidiosa, providing a more environmentally friendly and socially acceptable alternative to the use of chemical pesticides.

[0038] In the present invention, the terms “control of X. fastidiosa” or “biocontrol of X. fastidiosa,” used interchangeably, refer to the inhibition of biofilm formation, reduction, prevention of accumulation, and/or elimination of X. fastidiosa in a plant and/or in an insect vector of X. fastidiosa. Control of X. fastidiosa in an insect vector decreases transmission between plants. Therefore, control of X. fastidiosa in a plant and/or insect vector allows for the treatment and/or prevention of an infection caused by X. fastidiosa in the plant.

[0040] Xylella fastidiosa is a plant pathogenic bacterium native to the California region of the United States. Transmission of X. fastidiosa between host plants occurs through xylem-feeding insects (e.g., hemipteran insects of the family Cercopidae). Once introduced into the plant, X. fastidiosa colonizes the plant's xylem, causing vessel obstruction, leaf burn, and ultimately plant death.

[0042] Xylella fastidiosa is capable of infecting a wide variety of plants. Thus, in the present invention, the term “plant(s)” refers to any living organism belonging to the kingdom Plantae (i.e., any genus/species of the plant kingdom), where such organism is susceptible to being, or is, infected by the bacterium X. fastidiosa. Likewise, the term “plant” encompasses plants that have been modified by one or more of the methods of reproduction, mutagenesis, and genetic engineering, as well as any plant propagule.

[0044] The control of X. fastidiosa is of particular interest in crop plants since this pathogen causes large plant losses and therefore economic losses in the agricultural sector.

[0046] Thus, in a preferred embodiment of the use of the invention, the plant is a cultivated plant.

[0048] In the present invention, the term “cultivated plant” refers to any plant that is cultivated by humans for ornamental purposes or to obtain a product of interest from it (for example, to obtain fruits, tubers or a part of the plant such as the flower or its leaves).

[0050] Thus, in a preferred embodiment of the use of the invention, the plant is selected from the list consisting of olive, rice, apple, wheat, kiwi, corn, barley, millet, sorghum, oats, soybean, rye, alfalfa, sunflower, tobacco, potato, cotton, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, papaya, cashew, macadamia, almond, sugar beet, sugar cane, tomato, lettuce, pea, cucurbits, cherry, vine, conifers, and grass.

[0052] Plants susceptible to infection by X. fastidiosa can be inoculated with this bacterium by insect vectors such as hemipterans, which feed on the xylem of the plant. Therefore, transmission between plants occurs when an insect vector feeds on a plant infected with X. fastidiosa and subsequently feeds on a second plant, which then becomes infected. In the present invention, the terms “insect vector” or “X. fastidiosa vector insect,” used interchangeably, refer to an insect that acts as a vehicle for the transmission of X. fastidiosa between plants. The main insect vectors of X. fastidiosa between plants are hemipterans.

[0054] Thus, in a preferred embodiment of the use of the invention, the vector insect is a hemipteran.

[0055] Examples of hemipterans that act as vectors of X. fastidiosa in plants include, but are not limited to, hemipterans belonging to the families Cicadellidae, Cercopoidae, Aphrophoridae, or Cicadidae. Non-limiting examples of hemipteran vectors of X. fastidiosa include, but are not limited to, Homalodisca vitripennis, Philaenus spumarius, or Neophilaenus campestris.

[0057] In a more preferred embodiment of the use of the invention, the insect vector is a hemipteran belonging to a family selected from among Cicadellidae, Cercopididae, Aphrophoridae, and Cicadae.

[0059] In another preferred embodiment of the use of the invention, X. fastidiosa is selected from X. fastidiosa subsp. fastidiosa CECT 9898 and Xylella fastidiosa subsp. multiplex CECT 9899.

[0061] As a person skilled in the art knows, compounds suitable for use in agriculture for pest control may be included in a composition that also comprises other compounds acceptable in agriculture. In the present invention, the term “compound(s) acceptable in agriculture” refers to all compounds suitable for use in agriculture, where, in general, such compounds are not toxic to humans or the plant to which they are applied and, preferably, are environmentally friendly. Examples of compounds acceptable in agriculture include, without limitation, insect attractants, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, stabilizers, antioxidants, osmotic protectants, or sunscreens.

[0063] In a preferred embodiment of the use of the invention, the composition iii) comprising the bacterial strain belonging to the Bacillacea family and/or the supernatant of a culture thereof, further comprises one or more compounds acceptable in agriculture.

[0065] In a further preferred embodiment of the use of the invention, one or more compounds acceptable in agriculture are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens, and any combination thereof.

[0067] As the person skilled in the subject knows, compositions intended for use in agriculture may be formulated in liquid or solid form, including, without limitation, formulations in the form of granules, suspension concentrate or powder, depending on the type of crop, the plant or the route of administration to the plant.

[0069] Thus, in a preferred embodiment of the use of the invention, the composition iii) comprising the strain belonging to the Bacillacea family and/or the supernatant of a culture thereof, is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate or emulsifiable concentrate.

[0071] In another aspect, the present invention relates to a method for controlling X. fastidiosa in a plant, hereinafter referred to as the “plant control method of the invention,” wherein said method comprises applying to the whole plant, to a part of the plant, or to the soil where the plant is grown:

[0073] i) a bacterial strain belonging to the family Bacillaceae,

[0074] ii) a supernatant from a culture of a bacterial strain belonging to the family Bacillaceae, or

[0075] iii) a composition comprising i) and/or ii),

[0077] wherein the bacterial strain belonging to the Bacillaceae family in i) and ii) is selected from the list consisting of: Bacillus mojavensisCECT 30857, Bacillus velezensisCECT 30858, Bacillus subtilisCECT 30859, Peribacillus simplexCECT 30856 and Bacillus mycoidesCECT 30855.

[0079] The terms “plant” and “control of X. fastidiosa” have been described above in the use of the invention, and those terms are applicable in the present aspect as well as in its preferred embodiments.

[0081] In a preferred embodiment of the plant control method of the invention, i), ii) or iii) are applied in an effective amount.

[0083] In the present invention, the term “effective quantity” refers to the quantity of strain belonging to the family Bacillaceae (Bacillus velezensis CECT 30858, Bacillus subtilis CECT 30859, Bacillus mojavensis CECT 30857, Peribacillus simplex CECT 30856, Bacillus mycoides CECT 30855) or the supernatant of a culture thereof, which, when applied to the plant or ingested by an insect vector, produces the intended effect without producing a toxic effect. In the context of the present invention, the intended effect is the control of X. fastidiosa in a plant and/or in an insect vector. As is evident to a person skilled in the art, effective quantities may vary depending on several factors, such as, for example, (a) the type of plant or insect; (b) the physiological condition of the plant or insect; (c) the concentration of pathogens affecting the plant or agricultural product; (d) the type of disease affecting the plant or agricultural product; (e) the weather conditions (e.g., temperature, humidity); and (f) the stage of the plant disease. The expert in the field knows how to calculate the effective amount to be applied based on the factors mentioned.

[0085] In the context of the present invention, the term "apply" refers to bringing into contact (i) the strain of the Bacillaceae family, (ii) the supernatant of a culture thereof or the composition of one of the foregoing, with the plant, a part of the plant, or the soil where the plant is grown.

[0087] In a preferred embodiment of the plant control method of the invention, the plant part is selected from the list consisting of a leaf, a stem, a callus, a flower, a root, a tuber, and a seed.

[0089] In another preferred embodiment of the plant control method of the invention, the plant is a crop plant. The term “crop plant” has been described above in the use of the invention, and that term is applicable to the method of the invention.

[0091] In a more preferred embodiment of the plant control method of the invention, the plant is selected from the list consisting of: olive, rice, apple, wheat, kiwi, corn, barley, millet, sorghum, oats, soybeans, rye, alfalfa, sunflower, tobacco, potato, cotton, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, papaya, cashew, macadamia, almond, sugar beet, sugar cane, tomato, lettuce, pea, cucurbits, conifers, cherry, grapevine, and turf.

[0093] In another preferred embodiment of the plant control method of the invention, the composition iii) comprising the strain belonging to the Bacillacea family and/or the supernatant of a culture thereof, further comprises one or more compounds acceptable in agriculture.

[0094] In a further preferred embodiment of the plant control method of the invention, one or more agriculturally acceptable compounds are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens, and any combination thereof.

[0096] In another preferred embodiment of the plant control method of the invention, the insect is a hemipteran, more preferably a hemipteran belonging to a family selected from among Cicadellidae, Cercopididae, Aphrophoridae and Cicadae.

[0098] In another preferred embodiment of the plant control method of the invention, X. fastidiosa is selected from X. fastidiosa subsp. fastidiosa CECT 9898 and Xylella fastidiosa subsp. multiplex CECT 9899.

[0100] In another preferred embodiment of the plant control method of the invention, composition iii) comprising the strain belonging to the Bacillacea family and/or the supernatant of a culture thereof, is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate or emulsifiable concentrate.

[0102] In the plant control method of the invention, the application to the whole plant, to a part of the plant, or to the soil where the plant is grown, can be carried out by any known technique, such as, but not limited to, through hydroponics, drip irrigation, spraying, infiltration, injection, misting, coating, or fumigation.

[0104] In another aspect, the present invention relates to a composition, hereinafter referred to as the “composition of the invention”, comprising:

[0106] i) a bacterial strain belonging to the family Bacillaceae, and/or

[0107] ii) a supernatant from a culture of a bacterial strain belonging to the family Bacillaceae,

[0109] wherein the bacterial strain belonging to the Bacillaceae family in i) and ii) is selected from the list consisting of: Bacillus mojavensisCECT 30857, Bacillus velezensisCECT 30858,Bacillus subtilisCECT 30859,Peribacillus simplexCECT 30856,Bacillus mycoidesCECT 30855.

[0111] In a preferred embodiment of the “composition of the invention”, said composition further comprises one or more compounds acceptable in agriculture.

[0113] In a preferred embodiment of the composition of the invention, one or more compounds acceptable in agriculture are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens, and any combination thereof; more preferably the composition of the invention comprises an insect attractant.

[0115] In another preferred embodiment of the composition of the invention, said composition is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate, or emulsifiable concentrate.

[0117] In another preferred embodiment, the composition of the invention comprises an effective amount of:

[0118] i) the bacterial strain belonging to the Bacillaceae family, and/or

[0119] ii) a supernatant from a culture of a bacterial strain belonging to the family Bacillaceae.

[0121] In another preferred embodiment of the invention, the composition is an insect bait.

[0123] The bacterial strain or the supernatant of a culture of a bacterial strain belonging to the Bacillacea family or a composition comprising the foregoing, may be comprised in a device which can be placed in an environment comprising an insect vector of X. fastidiosa.

[0125] Thus, in another aspect, the present invention relates to a device, hereinafter the “device of the invention”, comprising at least one container, characterized in that the container comprises:

[0127] i) a bacterial strain belonging to the family Bacillaceae,

[0128] ii) a supernatant from a culture of a bacterial strain belonging to the family Bacillaceae, or

[0129] iii) a composition comprising i) and/or ii),

[0131] wherein the bacterial strain belonging to the Bacillaceae family in i) and ii) is selected from the list consisting of: Bacillus mojavensisCECT 30857, Bacillus velezensisCECT 30858, Bacillus subtilisCECT 30859, Peribacillus simplexCECT 30856 and Bacillus mycoidesCECT 30855.

[0133] In a preferred embodiment, the device of the invention is configured so that an insect vector can access the container of said device and/or the contents of said container; preferably the device and/or container comprises an opening of sufficient dimensions to allow an insect vector access to its contents.

[0135] In another preferred embodiment, the material from which the device of the invention is made is plastic, crystal, glass, cardboard, metal and/or paper.

[0137] In another preferred embodiment of the device of the invention, composition iii) further comprises one or more compounds acceptable in agriculture; more preferably one or more compounds acceptable in agriculture are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens and any combination thereof.

[0139] In another preferred embodiment of the device of the invention, composition iii) is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate, or emulsifiable concentrate.

[0141] In a more preferred embodiment of the device of the invention, composition iii) is the composition of the invention.

[0143] In another preferred embodiment, the device of the invention is an insect trap.

[0145] In another aspect, the present invention relates to the use of the device of the invention for the control of X. fastidiosa in an insect vector.

[0146] In a preferred embodiment of the use of the device of the invention, the vector insect is a hemipteran.

[0148] In another preferred embodiment of the use of the device of the invention, the vector insect is a hemipteran belonging to a family selected from among Cicadellidae, Cercopididae, Aphrophoridae and Cicadae.

[0150] In another preferred embodiment of the use of the device of the invention, X. fastidiosa is selected from X. fastidiosa subsp. fastidiosa CECT 9898 and Xylella fastidiosa subsp. multiplex CECT 9899.

[0152] In another aspect, the present invention relates to a method of controlling X. fastidiosa in an insect vector, hereinafter referred to as the “inseed vector control method of the invention”, comprising placing the composition of the invention or the device of the invention in an environment where the insect vector is found.

[0154] The expression “environment where the insect vector is found” refers to any environment, closed or open, in which an insect vector of X. fastidiosa is found and where it performs at least some of its biological functions such as feeding, growing and/or reproducing. The environment in which an insect vector of X. fastidiosa is found may be, without limitation, a field in which crops or ornamental plants are grown.

[0156] Thus, in a preferred embodiment of the control method in an insect of the invention, the environment a plant susceptible to, or suffering from, an infection caused by X. fastidiosa transmitted by the insect vector.

[0158] In another preferred embodiment of the insect control method of the invention, wherein the insect vector is a hemipteran.

[0160] In another preferred embodiment of the insect control method of the invention, the insect vector is a hemipteran belonging to a family selected from Cicadellidae, Cercopididae, Aphrophoridae, and Cicadidae.

[0162] In another preferred embodiment of the insect control method of the invention,X.

[0163] fastidiosa is selected from X. fastidiosasubsp.fastidiosaCECT 9898 and Xylella fastidiosasubsp.multiplexCECT 9899.

[0165] The uses, methods, and composition with the device of the invention also include bacteria derived from the strains Bacillus velezensis CECT 30858, Bacillus subtilis CECT 30859, Bacillus mojavensis CECT 30857, Peribacillus simplex CECT 30856, and Bacillus mycoides CECT 30855, which retain antimicrobial capacity against X. fastidiosa and therefore retain the ability to control this pathogenic bacterium in a plant. The bacteria derived from the strains mentioned in this paragraph may be mutants that exhibit variations in their genome compared to the genome of the strain of the invention. In this document, the terms “mutant” or “derivative”, used interchangeably, refer to any microorganism resulting from a mutation or modification in the DNA of an organism that results in a character (phenotype) not found in the wild type (“wild type” or “wt”) strain and that maintains antimicrobial characteristics against X. fastidiosa.

[0167] DESCRIPTION OF THE FIGURES

[0169] Fig. 1. Antimicrobial activity of culturable isolates of olive sporobiota against X. fastidiosa subsp. fastidiosa CECT 9898 and X. fastidiosa subsp. multiplex CECT 9899. Control: Saline solution (0.85% w/V); A) Bacillus velezensis CECT 30858; B) Bacillus subtilis CECT 30859; C) Bacillus mojavensis CECT 30857; D) Peribacillus simplex CECT 30856; E) Bacillus mycoides CECT 30855.

[0171] Fig. 2. Antimicrobial activity of supernatant of culturable isolates of olive sporobiota against X. fastidiosa subsp. fastidiosa CECT 9898 and X. fastidiosa subsp. multiplex CECT 9899. Control: Saline solution (0.85% w/V); A) Bacillus velezensis CECT 30858; B) Bacillus subtilis CECT 30859; C) Bacillus mojavensis CECT 30857; D) Peribacillus simplex CECT 30856; E) Bacillus mycoides CECT 30855.

[0173] EXAMPLES

[0175] The invention will now be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

[0176] Example 1. Isolation of bacteria from the olive sporobiota.

[0178] A total of 417 bacteria were isolated from soil, leaf surface (epiphyte), and leaf interior (endophyte) samples from olive groves in five different locations in Andalusia, Spain: Linares (LIN), Jimena (JP), Bedmar y Garciez (BG), La Guardia de Jaén (LGJ), and Málaga (MA). Subsequently, 45 were selected based on their safety profiles and established use in agricultural settings to evaluate their antagonistic activity against X. fastidiosa. The bacteria selected for antimicrobial activity against X. fastidiosa belonged to the family Bacillaceae, specifically the genera Peribacillus (Peribacillus simplex), Bacillus (Bacillus endophyticus, Bacillus thuringiensis, Bacillus subtilis, Bacillus velenzensis, Bacillus mycoydes), and Priesta (Priesta aryabhattai, Priesta megaterium).

[0180] Example 2. Antimicrobial activity of olive sporobiota bacteria against Xylella fastidiosa.

[0182] The antagonistic activity of the strains isolated from the sources in Example 1 was carried out by the double culture method on solid media. The strains Xylella fastidiosa subsp. fastidiosa with CECT deposit number 9898 and Xylella fastidiosa subsp. multiplex with CECT deposit number 9899 were cultured on PD3 medium for 5–7 days. After X. fastidiosa was cultured in triplicate on PD3 plates, and after 24 h, 2.5 μl of a suspension of the selected sporobiota isolate or soil extract in 0.85% (w/v) saline at 0.5 McFarland was added to the top of each lane. Antagonistic activity was evaluated after an additional incubation at 28°C for 7–10 days, visible as growth inhibition in the form of a halo around the sporobiota culture (Fig. 1). Table 1 describes the results obtained for those bacterial strains from Example 1 that showed antimicrobial activity against X. fastidiosa.

[0183] Table 1. List of cultivable olive sporobiota isolates with antimicrobial activity against X. fastidiosa subsp. fastidiosa CECT 9898 and X. fastidiosa subsp. multiplex CECT 9899.

[0185]

[0188] *The relative location of the candidate is indicated, as well as the mean halo size with its standard deviation (SD). Values with different letters are statistically different with P < 0.05 as determined by one-way analysis of variance (ANOVA) followed by Tukey's test.;[0190] Example 3. Antimicrobial activity of culture supernatant of olive sporobiota bacteria against Xylella fastidiosa.;[0192] Those cultivable isolates of olive sporobiota that showed antagonistic effects in dual culture assays were further evaluated for their ability to produce antimicrobial compounds against X. fastidiosa in broth. As before, all experiments were performed against X. fastidiosa subsp. fastidiosa CECT 9898 and X. fastidiosa subsp. multiplex CECT 9899. For this purpose, a fresh colony of the antimicrobial-producing strain to be tested was inoculated into 20 ml of PD3 and cultured at 37 °C for 3 days. The cells were then removed by centrifugation at 4000 rpm for 10 minutes, followed by filtration through a 0.22 μm filter. X. fastidiosa was cultured in triplicate on PD3 plates as described above and incubated overnight at 28 °C. The following day, a well was created at the top of each lane of X. fastidiosa culture and filled with 75 μl of filtered broth. Antimicrobial activity was assessed based on the inhibition of X. fastidiosa growth in the vicinity of the culture filtrate after 7–10 days of incubation at 28 °C (Fig. 2).*

Claims (37)

1. CLAIMS 1. Bacillus mycoides strain with CECT deposit number 30855 with antimicrobial activity against Xylella fastidiosa. 2. Use of: i) the bacterial strain of the preceding claim ii) the supernatant from a culture of a bacterial strain of the preceding claim, or iii) a composition comprising i) and/or ii), for the control of Xylella fastidiosa in a plant and/or in an insect vector. 3. Use according to claim 2, wherein the plant is a cultivated plant. 4. Use according to the preceding claim, wherein the plant is selected from the list consisting of: olive, rice, apple, wheat, kiwi, corn, barley, millet, sorghum, oats, soybean, rye, alfalfa, sunflower, tobacco, potato, cotton, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, papaya, cashew, macadamia, almond, sugar beet, sugar cane, tomato, lettuce, pea, cucurbits, conifers, cherry, vine, and turf. 5. Use according to claim 2, wherein the insect vector is a hemipteran. 6. Use according to the preceding claim, wherein the insect vector is a hemipteran belonging to a family selected from among Cicadellidae, Cercopididae, Aphrophoridae and Cicadae. 7. Use according to any one of claims 2 to 6, wherein X. fastidiosa is selected from X. fastidiosa subsp. fastidiosa CECT9898 and Xylella fastidiosa subsp. Multiplex CECT9899. 8. Use according to any one of claims 2 to 7, wherein composition iii) further comprises one or more compounds acceptable in agriculture. 9. Use according to the preceding claim, wherein one or more compounds acceptable in agriculture are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens and any combination thereof. 10. Use according to any one of claims 2 to 9, wherein composition iii) is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate or emulsifiable concentrate. 11. Method for the control of Xylella fastidiosa in a plant, wherein said method comprises applying to the whole plant, to a part of the plant or to the soil where the plant is grown: i) the bacterial strain of claim 1, ii) the supernatant from a culture of the bacterial strain of claim 1, or iii) a composition comprising i) and/or ii). 12. Method according to the preceding claim, wherein the plant part is selected from the list consisting of a leaf, a stem, a callus, a flower, a root, a tuber, and a seed. 13. Method according to any one of claims 11 or 12, wherein the plant is a cultivated plant. 14. Method according to the preceding claim, wherein the plant is selected from the list consisting of: olive, rice, apple, wheat, kiwi, corn, barley, millet, sorghum, oats, soybean, rye, alfalfa, sunflower, tobacco, potato, cotton, sweet potato, cassava, coffee, coconut, pineapple, citrus, cocoa, tea, banana, avocado, fig, guava, mango, papaya, cashew, macadamia, almond, sugar beet, sugar cane, tomato, lettuce, pea, cucurbits, conifers, cherry, vine, and grass. 15. Method according to any one of claims 11 to 14, wherein composition iii) further comprises one or more compounds acceptable in agriculture. 16. Method according to the preceding claim, wherein one or more compounds acceptable in agriculture are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens and any combination thereof. 17. Method according to any one of claims 11 to 16, wherein X. fastidiosa is selected from X. fastidiosa subsp. fastidiosa CECT 9898 and Xylella fastidiosa subsp. multiplex CECT 9899. 18. Method according to any one of claims 11 to 17, wherein composition iii) is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate or emulsifiable concentrate. 19. A composition comprising: i) the bacterial strain of claim 1 and/or ii) the supernatant from a culture of the bacterial strain of claim 1, preferably where the composition also comprises one or more compounds acceptable in agriculture. 20. Composition according to the preceding claim, wherein one or more compounds acceptable in agriculture are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens and any combination thereof. 21. Composition according to any one of claims 19 or 20, wherein said composition is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate or emulsifiable concentrate. 22. Composition according to any one of claims 19 to 21, characterized in that it is insect bait. 23. A device comprising at least one container, characterized in that the container comprises: i) the bacterial strain of claim 1, ii) the supernatant from a culture of the bacterial strain of claim 1, or iii) a composition comprising i) and/or ii). 24. Device according to the preceding claim, wherein composition iii) further comprises one or more compounds acceptable in agriculture. 25. Device according to the preceding claim, wherein one or more compounds acceptable in agriculture are selected from the list consisting of insect attractants, stabilizers, osmotic protectants, inorganic nutrients, organic nutrients, wetting agents, buffering compounds, antioxidants, osmotic protectants, sunscreens and any combination thereof. 26. Device according to any one of claims 23 to 25, wherein composition iii) is formulated as a solid formulation, liquid formulation, granules, powder, wettable powder, suspension concentrate or emulsifiable concentrate. 27. Device according to any one of claims 23 to 26, wherein composition iii) is the composition according to claims 19 to 22. 28. Device according to any one of claims 23 to 27, wherein the device is an insect trap. 29. Use of the device according to any one of claims 23 to 28, for the control of X. fastidiosa in an insect vector. 30. Use according to the preceding claim wherein the insect vector is a hemipteran. 31. Use according to the preceding claim, wherein the insect vector is a hemipteran belonging to a family selected from among Cicadellidae, Cercopididae, Aphrophoridae and Cicadae. 32. Use according to any one of claims 29 to 31, wherein X. fastidiosa is selected from X. fastidiosa subsp. fastidiosa CECT 9898 and Xylella fastidiosa subsp. multiplex CECT 9899. 33. A method for controlling X. fastidiosa in an insect vector comprising placing the composition according to any one of claims 19 to 22 or the device according to any one of claims 23 to 28, in an environment where the insect vector is found. 34. Control method according to the preceding claim, wherein the environment comprises a plant susceptible to, or suffering from, an infection caused by X. fastidiosa transmitted by the insect vector. 35. Method according to any one of claims 33 or 34, wherein the insect vector is a hemipteran. 36. Method according to any one of claims 33 to 35, wherein the insect vector is a hemipteran belonging to a family selected from Cicadellidae, Cercopididae, Aphrophoridae and Cicadidae. 37. Method according to any one of claims 33 to 36, wherein X. fastidiosa is selected from X. fastidiosa subsp. fastidiosa CECT 9898 and Xylella fastidiosa subsp. multiplex CECT 9899.